Effects of nitrogen reduction combined with bio-organic fertilizer on soil bacterial community diversity of red raspberry orchard

Understanding soil bacterial diversity under nitrogen reduction is necessary for the crucial role in soil nitrogen cycling. However, the effects of combined fertilization on soil chemical properties, microbial community structure, and yield are unknown. This study was conducted to investigate the effect of nitrogen fertilizer reduction with bio-organic fertilizer on soil bacterial community diversity of red raspberry orchard. Six treatments were set in this study: NF-100%, NF-75%, NF-50%, NF-25% and CF, no nitrogen fertilizer and bio-organic fertilizer for CK. The bacterial community structures of soil were analyzed by 16S rRNA gene amplification high-throughput sequencing technology. Nitrogen fertilizer reduction with bio-organic fertilizer increased soil organic matter (SOM), total nitrogen (TN), alkali-hydrolyzable nitrogen (AN), available phosphorus (AP), available potassium (AK), and reduced soil pH. NF-50% and NF-25% treatments increased the yield of red raspberry. Nitrogen reduction combined with bio-organic fertilizer increased the relative abundance of copiotrophic bacteria and decreased the relative abundance of oligotrophic bacteria. The increase in copiotrophic bacteria in the soil of red raspberry orchard could indicate an increase in soil nutrient availability, which have positive implications for soil fertility and production. However, nitrogen fertilizer reduction with bio-organic fertilizer altered the abundance and diversity of soil bacteria, which was reduced compared to CF treatments. The PCoA analysis of the soil bacterial community showed that the community structure of NF-25% treatment was more different from other treatments, indicating that the fertilization method changed the community structure of soil bacteria. The results of a redundancy analysis showed that SOM, pH, AN, TN, and AP were the main factors affecting the microbial community structure. Overall, the reduction of nitrogen fertilizer with bio-organic fertilizer significantly increased the soil nutrient content, reduced the relative abundance and diversity of soil bacteria, increased the relative abundance of beneficial bacteria in the soil, changed the bacterial community structure of soil, increased production and created suitable soil conditions for the red raspberry growth.


Introduction
Red raspberry (Rubus idaeusl L.) is an internationally popular third-generation fruit, which is rich in nutrients and contains anthocyanins, ellagic acid, salicylic acid, flavonoids and other active substances with anti-cancer [1], anti-aging [2], blood sugar [3] and lipid lowering effects [4,5]. Nitrogen fertilization is an important element that affects the yield of red raspberry [6]. In recent years, farmers have blindly invested excessive nitrogen fertilizer in agricultural production in pursuit of high yield and profitability, which has led to a series of problems such as soil consolidation, soil nutrient loss, decreasing utilization rate of nitrogen fertilizer, and affecting soil bacterial diversity and community structure stability [7]. Therefore, moderate reduction of nitrogen fertilizer application is an inevitable trend for the future development of raspberry industry.
Soil bacteria are the most abundant and active group of microorganisms that can effectively promote the decomposition and release nutrients and organic matter. It also plays an important role in soil nitrogen morphological conversion [8,9]. There are numerous microbial species in the soil, such as Azospirillum, Streptomyces, Bacillus, and Pseudomonas, which have been shown to have significant growth-promoting effects on plants, and soil-borne pathogens such as Fusarium, which can cause crop diseases [10]. The activity of soil bacteria is closely related to the physical and chemical properties of soil, and high application of nitrogen fertilizer would increase NO 3 -N content and decrease pH value in soil, which will affect the stability and diversity of soil bacterial community structure and change the supply capacity of effective soil nitrogen, ultimately affecting the growth and development of crops [11,12]. Biofertilizers, as substances containing living microorganisms, have been shown to improve soil fertility and crop yields and can be used as an alternative to chemical fertilizers [13]. Many studies have reported that chemical fertilizer reduction with bio-organic fertilizer can improve soil nutrient content, increase the abundance and diversity of soil bacteria, and change the soil bacterial community structure, thus promoting crop yield [14][15][16]. In recent years, most studies have focused on medicinal value [17], deep processing product development [18], and improving plant resistance [19], while few studies have been reported on the effect of nitrogen fertilizer reduction with bio-organic fertilizer on bacterial community diversity and structure in red raspberry soil. But the effects of bio-organic fertilizer and nitrogen application on soil nutrients and their relation to microbial properties was uncertain.
In this study, we investigated whether different gradients of nitrogen fertilizer reduction with bio-organic fertilizer could affect the physical and chemical properties and bacterial composition of red raspberry garden soil. We expect to provide a theoretical basis for maintaining soil bacterial diversity and clarifying the reasonable amount of nitrogen fertilizer application in the field to create suitable soil conditions for red raspberry growth.

Study site
The experiment was started in March 2021, and the test site was located in Sitai Village, Jingxiu District, Baoding City, Hebei Province, China (E115˚41 0 , N38˚84 0 ), which is 22 m above sea level with tidal soils. The test site belongs to the warm temperate semi-humid semi-arid monsoon climate zone, dry and windy in spring and autumn, hot and rainy in summer. The average annual rainfall is 550mm. The annual average temperature is 12.3˚C, and the highest and lowest temperatures are 41˚C and -23˚C, respectively. The soil organic matter (SOM), pH, total nitrogen (TN), alkali-hydrolyzable nitrogen (AN), available potassium (AK), and available phosphorus (AP) at a soil depth of 0-20 cm were 13.30 g kg -1 , 7.88, 1.01 g kg -1 , 9.05 mg kg -1 , 106.88 mg kg -1 , and 22.16 mg kg -1 , respectively.

Experimental design
Raspberries were planted in double-row strips in the trial site with 20 cm spacing between plants, 20 cm spacing between small rows and 2.5 m spacing between large rows. Double-season red raspberry "Polka" planted in 2019 was used as the test material. A total of six treatments with three replications of 3 m each (double rows), NF-100%: bio-organic fertilizer alone, NF-75%: conventional nitrogen application reduced by 75% + bio-organic fertilizer, NF-50%: conventional nitrogen application reduced by 50% + bio-organic fertilizer, NF-25%: conventional nitrogen application reduced by 25% + bio-organic fertilizer, CF: farmers' conventional nitrogen application (without bio-organic fertilizer), CK: no nitrogen and bioorganic fertilizer applied. The fertilizer application rates for different treatments are shown in Table 1. Bio-organic fertilizer was used as the base fertilizer and was applied in the way of strip ditches on both sides of the planting row before sprouting, with a dosage of 30 t/hm 2 . Nitrogen fertilizer (urea N46%) was applied in equal amounts at 10 cm of initial stem growth and one week before flowering (total 225 kg/hm 2 ). At 10 cm of primary stem growth, all treatments were fertilized with 120 kg/hm 2 each of phosphorus (monocalcium phosphate P 2 O 5 12%) and potassium (potassium sulphate K 2 O 50%) fertilizer. Bio-organic fertilizer: fermented sheep manure as carrier, containing 120 million/g of multifunctional microorganisms (Bacillus subtilis 100 million/g, Paenibacillus mucilaginosus 0.1 billion/g). Nutrient status: pH: 8.93, SOM: 183.99 g/kg, total nitrogen (TN): 6.6 g/kg, total phosphorus (TP): 5.95 g/kg, total potassium (TK): 16.09 g/kg, alkali-hydrolyzable nitrogen (AN): 1.43 g/kg, also contains zinc, magnesium, boron and other trace elements and potassium xanthate, etc.

Soil sampling and analysis
Destructive sampling was carried out on June 16, 2021, (bud stage) with three plots per treatment, and three red raspberry plants were randomly dug out whole from each plot, for a total of 54 plants. Soil samples from the same plot were evenly mixed and divided into two parts, one stored in a refrigerator at -80˚C for high-throughput sequencing analysis, and one taken back to the laboratory for natural drying and sieved through 1 mm for soil chemical property determination. Soil organic matter (SOM), pH, total nitrogen (TN), alkali-hydrolyzable nitrogen (AN), available phosphorus (AP), and available potassium (AK) were measured as described by Institute of Soil Science, Chinese Academy of Sciences [20].

Plant growth and yield
At the bud stage (June 16, 2021), three plants were selected from each plot for measurement, and 15 leaves from each plant were selected for leaf area measurement. The height and crown width of the red raspberry plants were measured with a tape measure and the ground diameter was measured with a vernier caliper. Leaf area was determined by scanning method, and the leaves were scanned using HP Scanjet 2400 scanner. The evaluation parameter was area yield

DNA extraction and 16S rRNA gene amplicon sequencing
Total genomic DNA samples were extracted using the OMEGA Soil DNA Kit (M5635 02) (Omega Bio Tek, Norcross, GA, USA), following the manufacturer's instructions, and stored at -20˚C prior to further analysis. The quantity and quality of extracted DNAs were measured using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. PCR amplification of the bacterial 16S rRNA genes V3-V4 region was performed using the forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR components contained 5 μl of buffer (5×), 0.25 μl of Fast pfu DNA Polymerase (5 U/μl), 2 μl (2.5 mM) of dNTPs, 1 μl (10 uM) of each Forward and Reverse primer, 1 μl of DNA Template, and 14.75 μl of ddH 2 O. Thermal cycling consisted of initial denaturation at 98˚C for 5 min, followed by 25 cycles consisting of denaturation at 98˚C for 30 s, annealing at 53˚C for 30 s, and extension at 72˚C for 45 s, with a final extension of 5 min at 72˚C. PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2×250 bp sequencing was performed using the Illlumina NovaSeq platform with NovaSeq 6000 SP Reagent Kit (500 cycles) at Shanghai Personal Biotechnology Co., Ltd (Shanghai, China).
Microbiome bioinformatics were performed with QIIME2 2019.4 with slight modification according to the official tutorials (https://docs.qiime2.org/2019.4/tutorials/tutorials/). Briefly, raw sequence data were demultiplexed using the demux plugin following by primers cutting with cutadapt plugin [21]. Sequences were then quality filtered, denoised, merged and chimera removed using the DADA2 plugin [22]. Non singleton amplicon sequence variants (ASVs) were aligned with mafft and used to construct a phylogeny with fasttree2. Alpha diversity metrics Chao1, Observed species, Shannon, Simpson, Faith's PD, Pielou's evenness and Good's coverage, beta dive rsity metrics weighted UniFrac, unweighted UniFrac, Jaccard distance, and Bray Curtis dissimilarity were estimated using the diversity plugin with samples. Taxonomy was assigned to ASVs using the classify sklearn na naïve Bayes taxonomy classifier in feature classifier plugin against the SILVA Release 132 Database.

Data analysis and processing
Excel 2010 software was used for data processing and graphing. SPSS 26.0 was used for data analysis, and One-way analysis of variance was performed using ANOVA (p < 0.05). Sequence data analyses were mainly performed using QIIME2 and R packages (v3.2.0). ASV-level alpha diversity indices, such as Chao1 richness estimator, Observed species, Shannon diversity index, Simpson index, Faith's PD, Pielou's evenness and Good's coverage were calculated using the ASV table in QIIME2, and visualized as box plots. ASV-level ranked abundance curves were generated to compare the richness and evenness of ASVs among samples. Beta diversity analysis was performed to investigate the structural variation of microbial communities across samples using UniFrac distance metrics [23,24] and visualized via principal coordinate analysis (PCoA). To further compare the differences in species composition between samples, the clustering results for each sample as well as for each taxonomic unit were calculated using the R script, and the abundance data for the top 50 genera in terms of mean abundance were used to create heat maps. LEfSe (Linear discriminant analysis effect size) was performed to detect differentially abundant taxa across groups using the default parameters [25]. The Redundancy analysis (RDA) and heat maps were performed by the genescloud tools, a free online platform for data analysis (https://www.genescloud.cn).

Soil physical and chemical properties
As shown in Table 2, nitrogen fertilizer reduction with bio-organic fertilizer had significant effects on soil chemical properties. the NF-25%, NF-50%, and NF-75% treatments significantly increased SOM, TN, AN, AP, and AK and decreased soil pH compared to CK. The content of SOM, TN, AN, AP and AK were much higher in the NF-25% treatment than in the other treatments. The results showed that nitrogen fertilizer reduction with bio-organic fertilizer could significantly improve soil fertility, and the best overall soil fertility effect was found in NF-25% treatment.

Soil bacterial diversity
Alpha diversity of the soil bacterial community under different fertilization treatments is shown in Fig 2. The Good's coverage index of all treatments was greater than 0.97, which also indicates that the sequencing ability can reflect the bacterial community characteristics of soil samples more accurately. CK of Chao1, Observed species index was the highest, followed by CF. Meanwhile, the Shannon and Simpson indices of CK treatment were higher than other treatments. Not coincidentally, the highest bacterial Pielou's evenness and Faith's PD indices were found in CK soil samples. It may be that bio-organic fertilizer contains a large number of beneficial active bacteria and organic matter, which can cause nutrient robbery among microorganisms, and the dominant population inhibits the reproduction of other populations.

Differential analysis of bacterial communities
PCoA analysis based on weighted-unifrac distance algorithm for soil samples with different fertilization treatments. As shown in Fig 3, the first principal component (PCo1) explains 63.3% of the variation in the bacterial community and the second principal component (PCo2) explains 10.7% of the variation. The closer the projection distance of the samples on the axes, the more similar the community composition of these two samples in the corresponding dimension. Similarly, the differences in community composition among CK, NF-50% and NF-100% were smaller. NF-25% was far from the other treatments, indicating that its bacterial community composition was more different from the other treatments.
To further explore the effect of nitrogen fertilizer reduction with bio-organic fertilizer on the soil community structure of red raspberry, cluster analysis was performed on the dominant soil bacteria at the genus level (Fig 4). CK, NF-100% and NF-50% were clustered into the same group, while NF-75% and CF were clustered into another group. The dominant genera of CK, NF-100% and NF-50% were Pedomicrobium, A4b and CCD24. In the other group, the dominant genera for NF-75% and CF were Bacillus, Streptomyces, JG30-KF-CM45, Sphingomonas and Actinoplanes. The dominant genera for NF-25% treatment was Allorhizobium-Neorhizobium-Parararhizobium-Rhizobium, Pseudoxanthomonas, Mycobacterium, Lysobacter, Luteimonas, Agromyces, and Pseudomonas, which differed significantly from the dominant genera in the other treatments.
LefSe linear discriminant analysis (LDA) was used to detect populations with significant differences in abundance between fertilization treatments (Fig 5). A total of six bacterial evolutionary branches showed significant differences in the different soil samples with an LDA threshold of 2. In CK, the relative abundance of Rhizobiales_Incertae_Sedis, Nordella was significantly higher than the other treatments. The relative abundance of Haliangiaceae, Haliangium was higher in the NF-100% treatment compared to the other treatments. NF-25% treatment of Faecalibaculum, Shinella was more enriched than other treatments.

Relationship between bacterial community structure and environmental factors
To investigate the effect of soil chemical properties on the bacterial community of red raspberry, RDA analysis was performed on the soil bacterial community and soil chemical

PLOS ONE
Effects of nitrogen reduction combined with bio-organic fertilizer on soil bacterial diversity properties of different fertilization treatments at the genus level. As shown in Fig 6, the two RDA components together explained 47.44% of the variation in bacterial community structure, with the first component (RDA1) explaining 7.70% of the variation; the second component (RDA2) explained 6.02% of the variation. As shown in Table 3, soil chemical properties

PLOS ONE
Effects of nitrogen reduction combined with bio-organic fertilizer on soil bacterial diversity had significant effects on soil bacterial community structure, with pH (P = 0.003), AN (P = 0.003), AP (P = 0.004) and SOM (P = 0.009) having highly significant effects (P < 0.01) on the bacterial community composition in the soil, and TN (P = 0.013) had a significant effect (P < 0.05) on the composition of the soil bacterial community.
Correlation analysis of major bacterial taxa and soil chemical properties in the soil of red raspberry garden, as shown in Fig 7. Subgroup_6, MND1, Rokubacteriales, bacteriap25, KD4 −96, RB41, Subgroup_17, Gaiella, 67−14 showed significant negative correlations (P < 0.05) with TN and SOM, highly significant negative correlations (P < 0.01) with AN, but significant positive correlated with pH (P < 0.05). Pseudomonas was significantly and positively correlated with TN, AP and SOM (P < 0.05), and highly significantly positively correlated with AN and AK (P < 0.01), but significantly negatively correlated with pH (P < 0.05).

Red raspberry plant growth and yield
As shown in Table 4, nitrogen fertilizer reduction with bio-organic fertilizer increased red raspberry plant height, ground diameter, crown size, and leaf area. Among them, NF-25% treatment had the most significant effect on promoting the growth of red raspberry trees.

PLOS ONE
Effects of nitrogen reduction combined with bio-organic fertilizer on soil bacterial diversity The effects of different fertilizer treatments on red raspberry yield are shown in Fig 8. Both NF-50% and NF-25% treatments increased the yield of red raspberry compared to CF treatment. The yield of red raspberry in NF-25% treatment was significantly higher than the other treatments, 5621.20 kg/hm 2 , which was 22.15% higher than CF. It showed that the reduction of nitrogen fertilizer with microbial organic fertilizer could significantly increase the yield of red raspberry. The lower overall yield of red raspberry may be due to the effect of long-term rainfall in the test site during the fruiting period, soil nutrient loss, and reduced photosynthetic efficiency of red raspberry, resulting in insufficient nutrient supply.

Discussion
In recent years, excessive application of nitrogen fertilizer in agricultural production has been widespread, leading to adverse effects such as crop yield reduction, decreased soil fertility, reduced soil enzyme activity, and reduced microbial community diversity [26,27]. Nitrogen fertilizer reduction with organic fertilizer not only increases yield, improves quality, and enriches soil but also improves soil quality, soil microbial diversity, and community structure [28,29]. In this study, nitrogen fertilizer reduction with bio-organic fertilizer treatment lowered soil pH and increased the content of soil organic matter, total nitrogen, alkali-hydrolyzable nitrogen, available phosphorus and available potassium contents, in which NF-25% treatment had the highest nutrient content. These results are consistent with the studies of Zhang [30] and He [31]. The reason is that the plant growth promoting rhinoacteria (PGPR) in bio-organic fertilizers have physiological functions such as phosphorus solubilization, potassium solubilization and nitrogen fixation, which release soluble potassium and phosphorus for crop uptake and utilization through certain pathways. It can also supply nitrogen by converting N 2 to NH 3 [32,33]. Application of bio-organic fertilizers enriches the soil with specific microorganisms, attracts beneficial bacteria, and promotes their reproduction, thus increasing soil nutrients.
In this study, the soil bacteria in the red raspberry garden mainly consisted of Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi and Bacteroidetes, which was consistent with the findings of Chen [34], Sun [35] and Zhang [36]. Proteobacteria plays an important role in the soil nitrogen cycle and helps to improve soil fertility and promote plant carbon and nitrogen uptake [37,38]. Acidobacteria has the role of promoting soil carbon cycle [39]. Campbell [40] et al. found a positive correlation between Acidobacteria and soil pH. In this study, the abundance of Proteobacteria increased significantly under nitrogen fertilizer reduction treatment, but the abundance of Acidobacteria decreased significantly. The reason was that nitrogen addition induces an increase in the relative abundance of soil Proteobacteria and a decrease in the relative abundance of Acidobacteria thus changing the community structure of soil bacteria, which were consistent with those of previous studies [11,41]. Actinobacteria have the ability to degrade mineral and organic matter and play a key role in promoting plant growth [42]. The differences in the relative abundance of Actinobacteria among fertilization treatments in this study were small. Studies have shown Proteobacteria and Bacteroidetes to be copiotrophic bacteria, Acidobacteria and Chloroflexi to be oligotrophic bacteria [43,44]. In this study, the relative abundance of Chloroflexi in NF-25% treatment was the lowest, and most of Chloroflexi were anaerobic bacteria, which means that the reduced abundance increased soil permeability and accelerated the rate of soil nutrient mineralization, thus facilitating nutrient uptake and utilization by the root system [45,46].
Soil bacterial diversity and richness were reduced in this study in the treatment of nitrogen fertilizer reduction with bio-organic fertilizer, which was inconsistent with the results of Shang [47]. and consistent with the results of Cai [48]. The main reason may be the additional application of bio-organic fertilizer on top of the nitrogen fertilizer reduction treatment, which contains a large amount of beneficial active bacteria and organic matter. It could cause nutrient robbery among microorganisms and the dominant population inhibited the reproduction of other populations [49]. The application of organic fertilizers introduces exogenous microorganisms that disrupt the dynamic balance of the original soil microorganisms and cause changes in the entire soil microbiota [50].
Differences in environmental factors can affect plant growth and development and the structure of their soil microbial communities [51,52]. Based on the results of this experiment, PCoA plots clearly distinguished the differences in soil bacterial microbial communities among fertilization treatments. These results were consistent with the findings of most longterm fertilization trials [53], in which application of different levels of N fertilizer with bioorganic fertilizer affected the soil microbial community structure to different degrees. In this study, LEfSe analysis and cluster analysis of the soil bacterial community revealed that the dominant genera differed significantly among treatments. CK and NF-100% were clustered into the same group, while NF-75%, CF and NF-50% were clustered into another group. However, the NF-25% treatment was significantly different from the dominant genus in the other treatments. The dominant genus of CK, Pedomicrobium, has the ability to retain iron and manganese in the soil environment, preventing the harmful effects of nutrient loss to plants [54]. Sphingomonas [55], a dominant genus in CF treatment, has a unique ability to degrade polycyclic aromatic hydrocarbons (PAHs), often present in petroleum-contaminated soils. In addition, studies have shown that Sphingomonas promote the growth of plants in saline areas [56]. It is possible that the soil of the test site is alkaline, and in the absence of nitrogen fertilization and organic fertilization, the red raspberry stimulated Sphingomonas to be in a dominant position in the soil in response to the unfavorable external conditions. Bacillus and Streptomyces are the dominant genera enriched in the NF-75% treatment. Bacillus has biocontrol function, while Bacillus and Streptomyces can promote the formation and stabilization of soil aggregates, reduce the evaporation of water from the soil surface, contribute to the reduction of soil salinity, and improve the environmental characteristics in the soil [57]. The dominant genus in the NF-25% treatment, Pseudomonas, has the ability to decomposing proteins, fats, and soil humus and which is a bacterium that promotes plant growth [58,59]. Luteimonas and Lysobacter are mercury-tolerant bacteria in soil and are resistant to mercury contamination [60]. Members of Shinella can effectively degrade micro-contaminants including polycyclic aromatic hydrocarbons (PAHs), among other micropollutants [61]. Anti-pollutant bacteria enrichment may be related to the application of nitrogen fertilizer, which increases the diversity of heavy metal-tolerant and nitrogen-fixing bacteria such as Pseudomonas, Azospirillum [62]. Studies have shown that nitrogen-fixing bacteria was tolerant to heavy metals and could remediate contaminated soil [63]. The addition of nitrogen fertilizer to external sources of petroleum polluted environment could promote the degradation of petroleum hydrocarbons. However, excessive application of nitrogen fertilizer not only caused an increase in nitrate content in the environment, resulting in secondary pollution, but also increased remediation costs [64,65]. Theoretically, if a significant proportion of the bacterial population was tolerant to a high concentration of metal contaminant, then the judgment was made that the soil was negatively affected by the presence of the metal. However, it had also been found that the number of heterotrophic nitrogen-fixing bacteria was significantly reduced in contaminated soil. Therefore, the reasons for the enrichment of such bacteria need to be further studied. The results of this study showed that nitrogen fertilizer reduction with bio-organic fertilizer could promote the decomposition of organic matter and soil pollutants and changed the community structure of soil bacteria.
Changes in soil chemistry under the influence of nitrogen fertilizer reduction with bioorganic fertilizer had different contributions to microbial taxa. In this study, key factors affecting the structure and composition of soil bacterial communities were revealed for pH, organic matter, total nitrogen, alkali-hydrolyzable nitrogen, and available phosphorus content. This was in general agreement with the results of Yang [66] and Zhou [67], who showed that soil pH, organic carbon and total nitrogen content significantly affected the community structure of bacteria. This indicates that the treatment of nitrogen fertilizer reduction with bio-organic fertilizer not only increased the nutrient content in the soil, but also affected the community composition of bacteria.
Microbial organic fertilizer affects the bacterial structure and abundance of the soil. The addition of microbial organic fertilizer provides sufficient substrate for soil microorganisms, which facilitates microbial colonization at the rhizosphere, leading to an increase in rhizosphere microbial diversity [68]. The large number of microorganisms carried by the exogenously added bioorganic fertilizer itself was also an important reason for the increased bacterial community diversity under the N fertilizer reduction treatment [69,70]. However, Li [71] found that the bacterial species carried by organic manure itself was significantly different from the composition of soil bacterial community after the application of chemical fertilizer combined with organic manure. Only a small percentage of the bacterial species in the soil samples came from the organic manure itself. Shi [72] found that Shannon diversity decreased, and bacterial community composition shifted in the bioorganic fertilizer treatment compared to non-fertilizer treatment. There was no significant difference in species richness between the two treatments. The addition of a bioorganic fertilizer also altered the metabolomic profile of rhizosphere soils and decreased the composition of many compounds belonging to the classes of phenolic acids, lipids, and flavonoids. Soil microorganisms mainly control most of the reactions in the nitrogen cycle [73]; Therefore, strong interactions between bacterial flora may occur after the addition of bioorganic fertilizers. A decrease in bacterial diversity might be due to plant response to changes in soil environmental conditions caused by bioorganic fertilization leading to the enrichment of a specific subset of functional microbiota in the rhizosphere [72]. At present, there were many researches [74][75][76] on the regulation of bioorganic fertilizer on soil bacterial diversity and structure, but there was lack of researches on the decrease of soil bacterial diversity caused by the application of bioorganic fertilizer. Therefore, this part will be the direction of our future research.

Conclusion
NF-25% treatment not only significantly increased the yield of red raspberry and the contents of soil organic matter, total nitrogen, alkali-hydrolyzable nitrogen, available phosphorus and available potassium, and reduced soil pH, but also increased the relative abundance of soil beneficial bacteria and changed the community structure of soil bacteria, and created soil environmental conditions suitable for the growth of red raspberry. However, the application of bioorganic fertilizer reduces soil bacterial richness and diversity, so we still need to conduct more research in the future to explore the combined benefits of bio-organic fertilizer application.